Skin UVB SKH1 mouse model treated with SFN
Part 1: RNA
Load RNA samples
Out of 30 samples, we selected 18 for this study. These are the normal tissue samples form the control, the UVA and the UVA+SFN treatment groups. normal tissue samples from the UVB_UA groups as well as tumor samples were excluded from this analysis.
First, we removed 7,148 genes with zero counts in > 80% (> 14 out of 18) of samples. 17,273 out of 24,421 genes left.
[1] 7148
[1] 17273Transcripts per kilobase million (TPM) normalization
Next, we noramized the counts. To convert number of hits to the relative abundane of genes in each sample, we used transcripts per kilobase million (TPM) normalization, which is as following for the j-th sample:
1. normilize for gene length: a[i, j] = 1,000*count[i, j]/gene[i, j] length(bp)
2. normalize for seq depth (i.e. total count): a(i, j)/sum(a[, j])
3. multiply by one million
A very good comparison of normalization techniques can be found at the following video:
RPKM, FPKM and TPM, clearly explained
After the normalization, each sample’s total is 1M:
02w_CON_0 02w_CON_1 02w_SFN_0 02w_SFN_1 02w_UVB_0 02w_UVB_1 15w_CON_0 15w_CON_1 15w_SFN_0 15w_SFN_1 15w_UVB_0 15w_UVB_1 25w_CON_0
1e+06 1e+06 1e+06 1e+06 1e+06 1e+06 1e+06 1e+06 1e+06 1e+06 1e+06 1e+06 1e+06
25w_CON_1 25w_SFN_0 25w_SFN_1 25w_UVB_0 25w_UVB_1
1e+06 1e+06 1e+06 1e+06 1e+06 Top 100 most abundant RNA molecules
# Separate top 100 abundant genes
tmp <- droplevels(tpm[Geneid %in% levels(tpm$Geneid)[(nrow(tpm) - 99):nrow(tpm)]])
tmp <- melt.data.table(data = tmp,
id.vars = 1:2,
measure.vars = 3:ncol(tmp),
variable.name = "Sample",
value.name = "TPM")
tmp$Week <- substr(x = tmp$Sample,
start = 1,
stop = 3)
tmp$Week <- factor(tmp$Week,
levels = unique(tmp$Week))
tmp$Treatment <- substr(x = tmp$Sample,
start = 5,
stop = 7)
tmp$Treatment <- factor(tmp$Treatment,
levels = c("CON",
"UVB",
"SFN"))
tmp$Replica <- substr(x = tmp$Sample,
start = 9,
stop = 9)
tmp$Replica <- factor(tmp$Replica,
levels = 0:1)
# Plot top 100 abundant genes
p2 <- ggplot(tmp,
aes(x = TPM,
y = Geneid,
fill = Treatment,
shape = Week)) +
# facet_wrap(~ Sex, nrow = 1) +
geom_point(size = 3,
alpha = 0.5) +
geom_vline(xintercept = 1,
linetype = "dashed")
ggplotly(p2)Bottom 100 least abundant RNA molecules
tmp <- droplevels(tpm[Geneid %in% levels(tpm$Geneid)[1:100]])
tmp <- melt.data.table(data = tmp,
id.vars = 1:2,
measure.vars = 3:ncol(tmp),
variable.name = "Sample",
value.name = "TPM")
tmp$Week <- substr(x = tmp$Sample,
start = 1,
stop = 3)
tmp$Week <- factor(tmp$Week,
levels = unique(tmp$Week))
tmp$Treatment <- substr(x = tmp$Sample,
start = 5,
stop = 7)
tmp$Treatment <- factor(tmp$Treatment,
levels = c("CON",
"UVB",
"SFN"))
tmp$Replica <- substr(x = tmp$Sample,
start = 9,
stop = 9)
tmp$Replica <- factor(tmp$Replica,
levels = 0:1)
# Plot top 100 abundant genes
p3 <- ggplot(tmp,
aes(x = TPM,
y = Geneid,
fill = Treatment,
shape = Week)) +
# facet_wrap(~ Sex, nrow = 1) +
geom_point(size = 3,
alpha = 0.5) +
geom_vline(xintercept = 1,
linetype = "dashed")
ggplotly(p3)Meta data
dmeta <- data.table(Sample = colnames(dt1)[-c(1:2)])
dmeta$time <- substr(x = dmeta$Sample,
start = 1,
stop = 3)
dmeta$time <- factor(dmeta$time,
levels = c("02w",
"15w",
"25w"))
dmeta$Week <- factor(dmeta$time,
levels = c("02w",
"15w",
"25w"),
labels = c("Week 2",
"Week 15",
"Week 25"))
dmeta$trt <- substr(x = dmeta$Sample,
start = 5,
stop = 7)
dmeta$trt <- factor(dmeta$trt,
levels = c("CON",
"UVB",
"SFN"))
dmeta$Treatment <- factor(dmeta$trt,
levels = c("CON",
"UVB",
"SFN"),
labels = c("Negative Control",
"Positive Control (UVB)",
"Sulforaphane (SFN)"))
dmeta$Replica <- substr(x = dmeta$Sample,
start = 9,
stop = 9)
dmeta$Replica <- factor(dmeta$Replica,
levels = 0:1)
datatable(dmeta,
options = list(pageLength = nrow(dmeta)))PCA of TPM
NOTE: the distributions are skewed. To make them symmetric, log transformation is often applied. However, there is an issue of zeros. In this instance, we added a small values lambda[i] equal to 1/10 of the smallest non-zero value of i-th gene.
dm.tpm <- as.matrix(tpm[, -c(1:2), with = FALSE])
rownames(dm.tpm) <- tpm$Geneid
# # Remove 02w_CON_1 sample and redo PCA
# dm.tpm <- dm.tpm[, colnames(dm.tpm) != "02w_CON_1"]
# dmeta <- dmeta[dmeta$Sample != "02w_CON_1", ]
# Add lambdas to all values, then take a log
dm.ltpm <- t(apply(X = dm.tpm,
MARGIN = 1,
FUN = function(a) {
lambda <- min(a[a > 0])/10
log(a + lambda)
}))
# PCA----
m1 <- prcomp(t(dm.ltpm),
center = TRUE,
scale. = TRUE)
s1 <- summary(m1)
s1Importance of components:
PC1 PC2 PC3 PC4 PC5 PC6 PC7 PC8 PC9 PC10 PC11 PC12
Standard deviation 70.7928 56.9107 50.8898 28.84564 26.51968 24.81005 23.85276 22.63644 20.97344 20.20442 19.24099 19.01279
Proportion of Variance 0.2901 0.1875 0.1499 0.04817 0.04072 0.03564 0.03294 0.02967 0.02547 0.02363 0.02143 0.02093
Cumulative Proportion 0.2901 0.4777 0.6276 0.67575 0.71647 0.75211 0.78504 0.81471 0.84018 0.86381 0.88524 0.90617
PC13 PC14 PC15 PC16 PC17 PC18
Standard deviation 18.73783 18.53642 17.87923 17.65132 17.16891 2.134e-13
Proportion of Variance 0.02033 0.01989 0.01851 0.01804 0.01707 0.000e+00
Cumulative Proportion 0.92650 0.94639 0.96490 0.98293 1.00000 1.000e+00Pareto chart of variance explained by principal components
imp <- data.table(PC = colnames(s1$importance),
Variance = 100*s1$importance[2, ],
Cumulative = 100*s1$importance[3, ])
imp$PC <- factor(imp$PC,
levels = imp$PC)
p1 <- ggplot(imp,
aes(x = PC,
y = Variance)) +
geom_bar(stat = "identity",
fill = "grey",
color = "black") +
geom_line(aes(y = rescale(Cumulative,
to = c(min(Cumulative)*30/100,
30)),
group = rep(1, nrow(imp)))) +
geom_point(aes(y = rescale(Cumulative,
to = c(min(Cumulative)*30/100,
30)))) +
scale_y_continuous("% Variance Explained",
breaks = seq(0, 30, by = 5),
labels = paste(seq(0, 30, by = 5),
"%",
sep = ""),
sec.axis = sec_axis(trans = ~.,
name = "% Cumulative Variance",
breaks = seq(0, 30, length.out = 5),
labels = paste(seq(0, 100, length.out = 5),
"%",
sep = ""))) +
scale_x_discrete("") +
theme(axis.text.x = element_text(angle = 90,
hjust = 1))
p1
First 3 principal components, pairwise
# Biplot while keep only the most important variables (Javier)----
# Select PC-s to pliot (PC1 & PC2)
choices <- c(1:3)
# Scores, i.e. points (df.u)
dt.scr <- data.table(m1$x[, choices])
# Add grouping variables
dt.scr$trt <- dmeta$trt
dt.scr$time <- dmeta$time
dt.scr$sample <- dmeta$Sample
# Loadings, i.e. arrows (df.v)
dt.rot <- as.data.frame(m1$rotation[, choices])
dt.rot$feat <- rownames(dt.rot)
dt.rot <- data.table(dt.rot)
# Axis labels
u.axis.labs <- paste(colnames(dt.rot)[choices],
sprintf('(%0.1f%% explained var.)',
100*m1$sdev[choices]^2/sum(m1$sdev^2)))
p1 <- ggplot(data = dt.scr,
aes(x = PC1,
y = PC2,
color = trt,
shape = time)) +
geom_point(size = 4,
alpha = 0.5) +
scale_x_continuous(u.axis.labs[1]) +
scale_y_continuous(u.axis.labs[2])
ggplotly(p1)
p1 <- ggplot(data = dt.scr,
aes(x = PC1,
y = PC3,
color = trt,
shape = time)) +
geom_point(size = 4,
alpha = 0.5) +
scale_x_continuous(u.axis.labs[1]) +
scale_y_continuous(u.axis.labs[3])
ggplotly(p1)
p1 <- ggplot(data = dt.scr,
aes(x = PC2,
y = PC3,
color = trt,
shape = time)) +
geom_point(size = 4,
alpha = 0.5) +
scale_x_continuous(u.axis.labs[2]) +
scale_y_continuous(u.axis.labs[3])
ggplotly(p1)First 3 principal components, 3D
scatterplot3js(x = dt.scr$PC1,
y = dt.scr$PC2,
z = dt.scr$PC3,
color = as.numeric(dt.scr$trt),
renderer = "auto",
pch = dt.scr$sample,
size = 0.1)Differential expressions
Session Information
sessionInfo()---
title: "Skin UVB SKH1 mouse model treated with SFN "
output:
  html_notebook:
    toc: yes
    toc_float: yes
    code_folding: hide
---

# Part 1: RNA
```{r header, echo = FALSE, message = FALSE, error = FALSE, warning  =FALSE}
# if (!requireNamespace("BiocManager", quietly = TRUE))
#     install.packages("BiocManager")
# BiocManager::install("DESeq2")

require(knitr)
require(data.table)
require(DT)
require(DESeq2)
require(readxl)
require(BiocParallel)
require(ggplot2)
require(plotly)
require(threejs)
require(scales)

# NOTE: on DESeq2 Output: 'baseMean' is the average of the normalized count values, 
# divided by the size factors, taken over all samples in the DESeqDataSet
```

## Load RNA samples
Out of 30 samples, we selected 18 for this study. These are the normal tissue samples form the control, the UVA and the UVA+SFN treatment groups. normal tissue samples from the UVB_UA groups as well as tumor samples were excluded from this analysis.     
First, we removed 7,148 genes with zero counts in > 80% (> 14 out of 18) of samples. 17,273 out of 24,421 genes left. 
         
```{r data_rna, warning = FALSE, echo = FALSE, message = FALSE}
# Load data----
dt0 <- fread("data/renyi_dedup_rnaseq_data/featurescounts_uvb-skin_dedup_renyi_2-9-2018.csv",
             skip = 1)

# Remove unused columns----
dt1 <- dt0[, c(1, 6:ncol(dt0)), with = FALSE]

cnames <- colnames(dt1)[-c(1:2)]
cnames <- gsub(x = cnames,
               pattern = ".dedup.bam",
               replacement = "")
colnames(dt1)[-c(1:2)] <- cnames

# ATTENTION! In this analysis, we will only examine controls and SFN
# Also, removed cancer cell samples
tnames <- substr(x = colnames(dt1), 
                 start = 3,
                 stop = 3)

gnames <- substr(x = colnames(dt1), 
                 start = 5,
                 stop = 7)

dt1 <- dt1[, gnames %in% c("id",
                           "th",
                           "CON",
                           "UVB",
                           "SFN" ) &
             tnames != "t",
           with = FALSE]
# 18 samples left

# Remove genes with zero counts in > 80% (> 14 out of 18) of samples
tmp <- dt1[, -c(1:2)] == 0
tmp <- rowSums(tmp) > 14
sum(tmp)

dt1 <- droplevels(dt1[!tmp, ])
nrow(dt1)
# 17,273 out of 24,421 genes left

datatable(head(dt1, 10),
              rownames = FALSE,
              options = list(pageLength = 10),
              caption = "Table 1: first 10 rows of the count table")
```

## Transcripts per kilobase million (TPM) normalization
Next, we noramized the counts. To convert number of hits to  the relative abundane of genes in each sample, we used ***transcripts per kilobase million (TPM)*** normalization, which is as following for the j-th sample:       
1. normilize for gene length: a[i, j] = 1,000*count[i, j]/gene[i, j] length(bp)     
2. normalize for seq depth (i.e. total count): a(i, j)/sum(a[, j])     
3. multiply by one million     
A very good comparison of normalization techniques can be found at the following video:    
[RPKM, FPKM and TPM, clearly explained](https://www.rna-seqblog.com/rpkm-fpkm-and-tpm-clearly-explained/)
     
After the normalization, each sample's total is 1M:
     
```{r tpm, warning = FALSE, echo = FALSE, message = FALSE}
# Normalize counts to TPM
tmp <- 1000*dt1[, 3:ncol(dt1)]/dt1$Length
tpm <- data.table(Geneid = dt1$Geneid,
                  Length = dt1$Length,
                  apply(tmp,
                        2,
                        function(a) {
                          10^6*(a/sum(a))
                        }))
colSums(tpm[, -c(1:2)])

formatRound(datatable(head(tpm, 10),
                      rownames = FALSE,
                      options = list(pageLength = 10),
                      caption = "Table 2: transcripts per kilobase million (TPM) normalized counts"),
            columns = 3:ncol(tpm),
            digits = 2)

# Total TPM
total <- rowSums(tpm[, 3:ncol(tpm)])

# Sort genes by relative abundancy
tpm$Geneid <- factor(tpm$Geneid ,
                     levels = tpm$Geneid[order(total,
                                               decreasing = FALSE)])
```

# Top 100 most abundant RNA molecules
```{r most_abundant}
# Separate top 100 abundant genes
tmp <- droplevels(tpm[Geneid %in% levels(tpm$Geneid)[(nrow(tpm) - 99):nrow(tpm)]])

tmp <- melt.data.table(data = tmp,
                       id.vars = 1:2,
                       measure.vars = 3:ncol(tmp),
                       variable.name = "Sample",
                       value.name = "TPM")

tmp$Week <- substr(x = tmp$Sample,
                   start = 1,
                   stop = 3)
tmp$Week <- factor(tmp$Week,
                   levels = unique(tmp$Week))


tmp$Treatment <- substr(x = tmp$Sample,
                        start = 5,
                        stop = 7)
tmp$Treatment <- factor(tmp$Treatment,
                        levels = c("CON", 
                                   "UVB",
                                   "SFN"))

tmp$Replica <- substr(x = tmp$Sample,
                      start = 9,
                      stop = 9)
tmp$Replica <- factor(tmp$Replica,
                      levels = 0:1)

# Plot top 100 abundant genes
p2 <- ggplot(tmp,
             aes(x = TPM,
                 y = Geneid,
                 fill = Treatment,
                 shape = Week)) +
  # facet_wrap(~ Sex, nrow = 1) +
  geom_point(size = 3,
             alpha = 0.5) +
  geom_vline(xintercept = 1,
             linetype = "dashed")
ggplotly(p2)
```

# Bottom 100 least abundant RNA molecules
```{r least_abundant}
tmp <- droplevels(tpm[Geneid %in% levels(tpm$Geneid)[1:100]])

tmp <- melt.data.table(data = tmp,
                       id.vars = 1:2,
                       measure.vars = 3:ncol(tmp),
                       variable.name = "Sample",
                       value.name = "TPM")

tmp$Week <- substr(x = tmp$Sample,
                   start = 1,
                   stop = 3)
tmp$Week <- factor(tmp$Week,
                   levels = unique(tmp$Week))


tmp$Treatment <- substr(x = tmp$Sample,
                        start = 5,
                        stop = 7)
tmp$Treatment <- factor(tmp$Treatment,
                        levels = c("CON", 
                                   "UVB",
                                   "SFN"))

tmp$Replica <- substr(x = tmp$Sample,
                      start = 9,
                      stop = 9)
tmp$Replica <- factor(tmp$Replica,
                      levels = 0:1)

# Plot top 100 abundant genes
p3 <- ggplot(tmp,
             aes(x = TPM,
                 y = Geneid,
                 fill = Treatment,
                 shape = Week)) +
  # facet_wrap(~ Sex, nrow = 1) +
  geom_point(size = 3,
             alpha = 0.5) +
  geom_vline(xintercept = 1,
             linetype = "dashed")
ggplotly(p3)
```

# Meta data
```{r meta}
dmeta <- data.table(Sample = colnames(dt1)[-c(1:2)])

dmeta$time <- substr(x = dmeta$Sample,
                   start = 1,
                   stop = 3)
dmeta$time <- factor(dmeta$time,
                   levels = c("02w",
                              "15w",
                              "25w"))
dmeta$Week <- factor(dmeta$time,
                   levels = c("02w",
                              "15w",
                              "25w"),
                   labels = c("Week 2",
                              "Week 15",
                              "Week 25"))

dmeta$trt <- substr(x = dmeta$Sample,
                        start = 5,
                        stop = 7)
dmeta$trt <- factor(dmeta$trt,
                        levels = c("CON", 
                                   "UVB",
                                   "SFN"))
dmeta$Treatment <- factor(dmeta$trt,
                        levels = c("CON", 
                                   "UVB",
                                   "SFN"),
                        labels = c("Negative Control",
                                   "Positive Control (UVB)",
                                   "Sulforaphane (SFN)"))

dmeta$Replica <- substr(x = dmeta$Sample,
                      start = 9,
                      stop = 9)
dmeta$Replica <- factor(dmeta$Replica,
                      levels = 0:1)

datatable(dmeta,
          options = list(pageLength = nrow(dmeta)))
```

# PCA of TPM
NOTE: the distributions are skewed. To make them symmetric, log transformation is often applied. However, there is an issue of zeros. In this instance, we added a small values ***lambda[i]*** equal to 1/10 of the smallest non-zero value of *i*-th gene. 
```{r pca}
dm.tpm <- as.matrix(tpm[, -c(1:2), with = FALSE])
rownames(dm.tpm) <- tpm$Geneid

# # Remove 02w_CON_1 sample and redo PCA
# dm.tpm <- dm.tpm[, colnames(dm.tpm) != "02w_CON_1"]
# dmeta <- dmeta[dmeta$Sample != "02w_CON_1", ]

# Add lambdas to all values, then take a log
dm.ltpm <- t(apply(X = dm.tpm,
                      MARGIN = 1,
                      FUN = function(a) {
                        lambda <- min(a[a > 0])/10
                        log(a + lambda)
                      }))

# PCA----
m1 <- prcomp(t(dm.ltpm),
             center = TRUE,
             scale. = TRUE)

s1 <- summary(m1)
s1
```

# Pareto chart of variance explained by principal components
```{r pca_var_plot}
imp <- data.table(PC = colnames(s1$importance),
                  Variance = 100*s1$importance[2, ],
                  Cumulative = 100*s1$importance[3, ])
imp$PC <- factor(imp$PC,
                 levels = imp$PC)
p1 <- ggplot(imp,
             aes(x = PC,
                 y = Variance)) +
  geom_bar(stat = "identity",
           fill = "grey",
           color = "black") +
  geom_line(aes(y = rescale(Cumulative,
                            to = c(min(Cumulative)*30/100,
                                   30)),
                group = rep(1, nrow(imp)))) +
  geom_point(aes(y = rescale(Cumulative,
                             to = c(min(Cumulative)*30/100,
                                    30)))) +
  scale_y_continuous("% Variance Explained",
                     breaks = seq(0, 30, by = 5),
                     labels = paste(seq(0, 30, by = 5),
                                    "%",
                                    sep = ""),
                     sec.axis = sec_axis(trans = ~.,
                                         name = "% Cumulative Variance",
                                         breaks = seq(0, 30, length.out = 5),
                                         labels = paste(seq(0, 100, length.out = 5),
                                                        "%",
                                                        sep = ""))) +
  scale_x_discrete("") +
  theme(axis.text.x = element_text(angle = 90,
                                   hjust = 1))
p1
```

# First 3 principal components, pairwise
```{r pca_plots}
# Biplot while keep only the most important variables (Javier)----
# Select PC-s to pliot (PC1 & PC2)
choices <- c(1:3)

# Scores, i.e. points (df.u)
dt.scr <- data.table(m1$x[, choices])
# Add grouping variables
dt.scr$trt <- dmeta$trt
dt.scr$time <- dmeta$time
dt.scr$sample <- dmeta$Sample

# Loadings, i.e. arrows (df.v)
dt.rot <- as.data.frame(m1$rotation[, choices])
dt.rot$feat <- rownames(dt.rot)
dt.rot <- data.table(dt.rot)

# Axis labels
u.axis.labs <- paste(colnames(dt.rot)[choices], 
                     sprintf('(%0.1f%% explained var.)', 
                             100*m1$sdev[choices]^2/sum(m1$sdev^2)))

p1 <- ggplot(data = dt.scr,
             aes(x = PC1,
                 y = PC2,
                 color = trt,
                 shape = time)) +
  geom_point(size = 4,
             alpha = 0.5) +
  scale_x_continuous(u.axis.labs[1]) +
  scale_y_continuous(u.axis.labs[2])
ggplotly(p1)

p1 <- ggplot(data = dt.scr,
             aes(x = PC1,
                 y = PC3,
                 color = trt,
                 shape = time)) +
  geom_point(size = 4,
             alpha = 0.5) +
  scale_x_continuous(u.axis.labs[1]) +
  scale_y_continuous(u.axis.labs[3])
ggplotly(p1)

p1 <- ggplot(data = dt.scr,
             aes(x = PC2,
                 y = PC3,
                 color = trt,
                 shape = time)) +
  geom_point(size = 4,
             alpha = 0.5) +
  scale_x_continuous(u.axis.labs[2]) +
  scale_y_continuous(u.axis.labs[3])
ggplotly(p1)
```

# First 3 principal components, 3D
```{r pca_3d, fig.height = 10, fig.width = 10}
scatterplot3js(x = dt.scr$PC1, 
               y = dt.scr$PC2, 
               z = dt.scr$PC3, 
               color = as.numeric(dt.scr$trt),
               renderer = "auto",
               pch = dt.scr$sample,
               size = 0.1)
```


# Differential expressions
```{r deseq2}

```


# Session Information
```{r info,eval=TRUE}
sessionInfo()
```